Toluidine blue O-induced photoinactivation inhibit the biofilm formation of methicillin-resistant Staphylococcus aureus.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to conventional antimicrobial therapies, allowing for high morbidity and mortality. Photodynamic antimicrobial chemotherapy (PACT) is one method that combines visible harmless light with the optimum wavelength with photosensitizers or dyes, producing singlet oxygen (1O2) and reactive oxygen strains (ROS), making permanent damages to the target cells. The purpose of this research is to evaluate the suppression efficacy of toluidine blue O (TBO)-mediated PACT on mature MRSA biofilm in vitro. METHODS: In this study, the 48 h mature biofilm of the multidrug-resistant Staphylococcus aureus strain MRSA252 was used. The photodynamic therapy (PDT) group was treated with different concentrations of TBO (0.5, 0.75, 1.0 or 1.25 µM) and different doses of red light (635 ± 5 nm wavelength; 30 or 50 J/cm2). The biofilms viability after PDT were evaluated by crystal violet (CV) staining assay and {2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetra-zolium hydroxide} (XTT) assay; meanwhile, the morphological changes were detected by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), separately. Moreover, the biofilms virulence was evaluated by red blood cell (RBC) hemolysis assay and staphylococcal virulence factor enterotoxins A (SEA) detected by enzyme linked immunosorbent assay (ELISA). After PDT, the biofilm was re-cultured for extra 48 h. Its formation viability and virulence were detected again. All data were analyzed by ANOVAs followed by the Games Howell post hoc test (α = 0.05). RESULTS: The biofilm was inactivated about 2.3 log10 at 1.25 µM with 30 J/cm2 illumination, and 3.5 log10 with 50 J/cm2 after PDT (P<0.05). XTT assays demonstrated the viability of mature MRSA biofilms was reduced after PACT. PDT group shows a distinct reduction in RBC hemolysis rate and the concentration of SEA compared to the control groups. The morphological features of the biofilms showed great changes, such as shrinkage, fissure, fragmentation, and rarefaction after being treated by TBO-PDT and observed by SEM. The recovery of the structure and virulence of biofilm were suppressed after PDT. CONCLUSION: TBO-mediated PDT could destroy the biofilm structure, reduce its virulence and depress its self-recovery.

Inhibition of efflux pump encoding genes and biofilm formation by sub-lethal photodynamic therapy in methicillin susceptible and resistant Staphylococcus aureus.

BACKGROUND: Photodynamic therapy (PDT) is an effective method to inactivate microorganisms which is based on reactive oxygen species (ROS) generated by photosensitizer and light at certain wavelength. Exposure to sub-lethal dose of PDT (sPDT) could activate the regulatory systems in the surviving bacteria in response to oxidative stress. This study aimed to evaluate the effect of sPDT on efflux pump and biofilm formation in Staphylococcus aureus (S. aureus), which are two important virulence related factors. METHODS: Different light irradiation time and toluidine blue O (TBO) concentrations were tested to select a sPDT in methicillin-susceptible and methicillin-resistant S. aureus (MSSA and MRSA). Efflux function was evaluated with EtBr efflux experiment. Biofilm formation was evaluated by crystal violet staining. Gene expressions of norA, norB, sepA, mepA and mdeA following sPDT were analyzed with real-time PCR. RESULTS: Sub-lethal PDT was set at 40 J/cm2 associated with 0.5 µM TBO. Efflux function was significantly inhibited in both strains. The average expression levels of mdeA and mepA in MSSA and MRSA were increased by (3.09, 1.77, 1.57) and (3,44, 1.59, 6.29) fold change respectively, norB and sepA were decreased by (3.77, 6.14) and (3.02, 3.47) fold change respectively. Expression level of norA was decreased by 5.44-fold change in MSSA but increased by 2.80-fold change in MRSA. Biofilm formation in both strains was impeded. CONCLUSIONS: TBO-mediated sPDT could inhibit efflux pump function, alter efflux pump encoding gene expression levels and retard biofilm formation in MSSA and MRSA. Therefore, sPDT is proposed as a potential adjuvant therapy for infections.

Photodynamic therapy accelerates skin wound healing through promoting re-epithelialization.

BACKGROUND: Epidermal stem cells (EpSCs) that reside in cutaneous hair follicles and the basal layer of the epidermis are indispensable for wound healing and skin homeostasis. Little is known about the effects of photochemical activation on EpSC differentiation, proliferation and migration during wound healing. The present study aimed to determine the effects of photodynamic therapy (PDT) on wound healing in vivo and in vitro. METHODS: We created mouse full-thickness skin resection models and applied 5-aminolevulinic acid (ALA) for PDT to the wound beds. Wound healing was analysed by gross evaluation and haematoxylin-eosin staining in vivo. In cultured EpSCs, protein expression was measured using flow cytometry and immunohistochemistry. Cell migration was examined using a scratch model; apoptosis and differentiation were measured using flow cytometry. RESULTS: PDT accelerated wound closure by enhancing EpSC differentiation, proliferation and migration, thereby promoting re-epithelialization and angiogenesis. PDT inhibited inflammatory infiltration and expression of proinflammatory cytokines, whereas the secretion of growth factors was greater than in other groups. The proportion of transient amplifying cells was significantly greater in vivo and in vitro in the PDT groups. EpSC migration was markedly enhanced after ALA-induced PDT. CONCLUSIONS: Topical ALA-induced PDT stimulates wound healing by enhancing re-epithelialization, promoting angiogenesis as well as modulating skin homeostasis. This work provides a preliminary theoretical foundation for the clinical administration of topical ALA-induced PDT in skin wound healing.